Bottom Line:
In general, contamination of surfaces by micro-organisms is a major source of problems in health care.Physical plasmas offer a huge potential to inactivate micro-organisms and to remove organic materials through plasma-generated highly reactive agents.The impact of plasma etching on biofilms is localized due to the limited presence of reactive plasma species validated by optical emission spectroscopy.

Introduction: The medical use of non-thermal physical plasmas is intensively investigated for sterilization and surface modification of biomedical materials. A further promising application is the removal or etching of organic substances, e.g., biofilms, from surfaces, because remnants of biofilms after conventional cleaning procedures are capable to entertain inflammatory processes in the adjacent tissues. In general, contamination of surfaces by micro-organisms is a major source of problems in health care. Especially biofilms are the most common type of microbial growth in the human body and therefore, the complete removal of pathogens is mandatory for the prevention of inflammatory infiltrate. Physical plasmas offer a huge potential to inactivate micro-organisms and to remove organic materials through plasma-generated highly reactive agents.

Method: In this study a Candida albicans biofilm, formed on polystyrene (PS) wafers, as a prototypic biofilm was used to verify the etching capability of the atmospheric pressure plasma jet operating with two different process gases (argon and argon/oxygen mixture). The capability of plasma-assisted biofilm removal was assessed by microscopic imaging.

Results: The Candida albicans biofilm, with a thickness of 10 to 20 µm, was removed within 300 s plasma treatment when oxygen was added to the argon gas discharge, whereas argon plasma alone was practically not sufficient in biofilm removal. The impact of plasma etching on biofilms is localized due to the limited presence of reactive plasma species validated by optical emission spectroscopy.

pone-0042539-g003: Comparison between Ar plasma and Ar/O2 plasma on etched surface area of 7-day old Candida albicans biofilms depending on the plasma treatment time (n = 5; mean ± SD).The biofilm-covered surface area was measured before and after plasma treatment. The control represents samples exposed to the gas flow without plasma ignition for 60 and 300 s. The initial surface area of the biofilm was 35171±3335 µm2 (n = 60). The right y-axis represents the percentage decrease of the biofilm-covered surface area.

Mentions:
The investigations demonstrate the removal or etching of biofilms by means of an atmospheric pressure plasma jet and extend our previous data, in which the efficacy of the plasma jet in killing, but not in removal of micro-organisms, was shown [25], [26]. Figure 1 shows representative images of C. albicans biofilms before and after 60 s (A) Ar/O2 gas flow (control), (B) Ar plasma, and (C) Ar/O2 plasma exposure. Initial removal - apparent as bright spots representing the PS subsurface - of C. albicans biofilm was observed after 60 s Ar plasma treatment (Fig. 1 (B)). Distinct biofilm removal, however, could be observed only with Ar/O2 plasma (Fig. 1 (C)). Figure 2 depicts a comparison of non-treated biofilm and biofilm exposed to (D) Ar and (E) Ar/O2 gas discharge plasma for 300 s. As can be seen in Fig. 2 D, longer Ar plasma exposure time did not result in substantial etching of the biofilm. In contrast, an almost complete removal of biofilm was obtained after Ar/O2 plasma treatment (Fig. 2 (E)). Consequently, to obtain increased biofilm removal, long time exposure was required. All control samples exposed to non-ionized Ar and Ar/O2 gases for 60 s and 300 s showed no biofilm etching. Hence, the process gas alone did not result in biofilm removal. To estimate the etching efficacy of the plasma jet, the biofilm-covered surface area of each sample was calculated before and after plasma exposure. The results of plasma-etched surface areas, i.e. the difference of the biofilm-covered area before and after exposure to gas discharge plasma, depending on treatment times and process gases, are displayed in Fig. 3. As previously noted, even longer treatment times with Ar plasma did not significantly increase the etched biofilm surface area. After 300 s Ar plasma exposure a maximum surface area of 5000 µm2 of initial 35171 µm2 (mean of the untreated biofilm-covered surface area) was etched. Compared to Ar plasma treatment, the admixture of oxygen increased the biofilm removal considerably, especially for treatment times above 60 s. Further shown in Fig. 3, is the percentage decrease of the biofilm (right y-axis). A reduction in biofilm-covered surface area of only 12% was attained with 300 s Ar plasma. In contrast, already after 180 s Ar/O2 plasma exposure, approximately 95% of the biofilm was etched. Consequently, the admixture of oxygen is crucial to obtain a sufficient efficacy in biofilm removal.

pone-0042539-g003: Comparison between Ar plasma and Ar/O2 plasma on etched surface area of 7-day old Candida albicans biofilms depending on the plasma treatment time (n = 5; mean ± SD).The biofilm-covered surface area was measured before and after plasma treatment. The control represents samples exposed to the gas flow without plasma ignition for 60 and 300 s. The initial surface area of the biofilm was 35171±3335 µm2 (n = 60). The right y-axis represents the percentage decrease of the biofilm-covered surface area.

Mentions:
The investigations demonstrate the removal or etching of biofilms by means of an atmospheric pressure plasma jet and extend our previous data, in which the efficacy of the plasma jet in killing, but not in removal of micro-organisms, was shown [25], [26]. Figure 1 shows representative images of C. albicans biofilms before and after 60 s (A) Ar/O2 gas flow (control), (B) Ar plasma, and (C) Ar/O2 plasma exposure. Initial removal - apparent as bright spots representing the PS subsurface - of C. albicans biofilm was observed after 60 s Ar plasma treatment (Fig. 1 (B)). Distinct biofilm removal, however, could be observed only with Ar/O2 plasma (Fig. 1 (C)). Figure 2 depicts a comparison of non-treated biofilm and biofilm exposed to (D) Ar and (E) Ar/O2 gas discharge plasma for 300 s. As can be seen in Fig. 2 D, longer Ar plasma exposure time did not result in substantial etching of the biofilm. In contrast, an almost complete removal of biofilm was obtained after Ar/O2 plasma treatment (Fig. 2 (E)). Consequently, to obtain increased biofilm removal, long time exposure was required. All control samples exposed to non-ionized Ar and Ar/O2 gases for 60 s and 300 s showed no biofilm etching. Hence, the process gas alone did not result in biofilm removal. To estimate the etching efficacy of the plasma jet, the biofilm-covered surface area of each sample was calculated before and after plasma exposure. The results of plasma-etched surface areas, i.e. the difference of the biofilm-covered area before and after exposure to gas discharge plasma, depending on treatment times and process gases, are displayed in Fig. 3. As previously noted, even longer treatment times with Ar plasma did not significantly increase the etched biofilm surface area. After 300 s Ar plasma exposure a maximum surface area of 5000 µm2 of initial 35171 µm2 (mean of the untreated biofilm-covered surface area) was etched. Compared to Ar plasma treatment, the admixture of oxygen increased the biofilm removal considerably, especially for treatment times above 60 s. Further shown in Fig. 3, is the percentage decrease of the biofilm (right y-axis). A reduction in biofilm-covered surface area of only 12% was attained with 300 s Ar plasma. In contrast, already after 180 s Ar/O2 plasma exposure, approximately 95% of the biofilm was etched. Consequently, the admixture of oxygen is crucial to obtain a sufficient efficacy in biofilm removal.

Bottom Line:
In general, contamination of surfaces by micro-organisms is a major source of problems in health care.Physical plasmas offer a huge potential to inactivate micro-organisms and to remove organic materials through plasma-generated highly reactive agents.The impact of plasma etching on biofilms is localized due to the limited presence of reactive plasma species validated by optical emission spectroscopy.

Introduction: The medical use of non-thermal physical plasmas is intensively investigated for sterilization and surface modification of biomedical materials. A further promising application is the removal or etching of organic substances, e.g., biofilms, from surfaces, because remnants of biofilms after conventional cleaning procedures are capable to entertain inflammatory processes in the adjacent tissues. In general, contamination of surfaces by micro-organisms is a major source of problems in health care. Especially biofilms are the most common type of microbial growth in the human body and therefore, the complete removal of pathogens is mandatory for the prevention of inflammatory infiltrate. Physical plasmas offer a huge potential to inactivate micro-organisms and to remove organic materials through plasma-generated highly reactive agents.

Method: In this study a Candida albicans biofilm, formed on polystyrene (PS) wafers, as a prototypic biofilm was used to verify the etching capability of the atmospheric pressure plasma jet operating with two different process gases (argon and argon/oxygen mixture). The capability of plasma-assisted biofilm removal was assessed by microscopic imaging.

Results: The Candida albicans biofilm, with a thickness of 10 to 20 µm, was removed within 300 s plasma treatment when oxygen was added to the argon gas discharge, whereas argon plasma alone was practically not sufficient in biofilm removal. The impact of plasma etching on biofilms is localized due to the limited presence of reactive plasma species validated by optical emission spectroscopy.